Cell differentiation in Dictyostelium requires that genetically identical cells follow alternate development pathways leading either to programmed cell death or to reinitiation of the life cycle. This process requires precise temporal and spatial expression of glycogen synthesis and degradation, to provide the precursor for the end products of terminal differentiation. In the proposed research, we will test several hypothesis regarding the molecular mechanisms of cell differentiation: (1) Transcriptional regulation of gp1 and gp2. We will utilize site- directed mutagenesis and fine deletion analysis of the glycogen phosphorylase (gp1 and gp2) promoters, to confirm the role of sequence elements in transcription regulation and for trans-acting factor recognition. We will clone the specific transcription factors (TF) by: 1) purifying the protein to obtain sequence information for use in PCR amplification and cloning, 2) employing a modification of the recently developed restriction enzyme-mediate integration technique (REMI), and 3) implementing a yeast genetic assay for the detection and cloning of gene products involved in protein-protein interactions. To determine the developmental significance of the trans-acting factors, cloned TF fragment will be used to alter the normal expression of that factor in Dictyostelium cells. (2) Signal transduction pathways. Cyclic AMP regulation of gp1, occurs through a mechanism involving intracellular cAMP signaling. Gp2 is regulated by transcription, through a pathway that involves cAMP surface receptors, but that is independent of intracellular cAMP. To identify the signal transduction pathways implicated in the regulation of gp1 and gp2, we will use several Dictyostelium mutant clones that fail to express either cAMP surface receptors, DIF, cAMP dependent protein kinase, adenylate cyclase, phospholipase C, or glycogen synthase kinase. (3) The significance of gp to cell differentiation. Double gp 1-2-mutant cell lines will serve as a "blank slat" for the re-introduction of gp1 or gp2 coding regions under the control of alternative Dictyostelium prestalk or prespore cell- specific promoters. This ability to express a phosphorylase gene in one cell type, but not the other, will allow a direct test of the extent to which the initiation of glycogen breakdown is limiting for cell-specific differentiation. (4) Cloning and characterization of the glycogen synthase gene. We will characterize the glycogen synthase gene and protein in terms of: 1) developmental and cell-type expression, 2) the factors that influence transcription (cAMP, DIF, etc.), and 3) the cis genomic regulatory elements, as we have already done for gp1 and gp2. Because of the unique division of aging and non-aging cells in this system, and the presence of genes that respond, accordingly, to molecules that are known to be regulators of the alternate pathways of cell differentiation, we believe that the proposed research will be successful in helping to define the regulatory pathways involved in programmed cell death.